Hemagglutination method for australia antigen and antibody thereto

ABSTRACT

Australia antigen or the antibody thereto is detected in the serum of a patient with viral hepatitis or detected in other appropriate biological material by a hemagglutination assay wherein indicator red cells are coated with purified hepatitisassociated-antigen and serum taken from the patient or other biological material is mixed therewith. If antibodies are present in the tested material hemagglutination will occur. In the test for the antigen the serum or other biological material is mixed with antibodies prior to contacting the mixture with the coated cells. Hemagglutination-inhibition indicates the presence of antigen in the test material.

United States Patent [1 1 Vyas et al.

[ June 3, 1975 1 HEMAGGLUTINATION METHOD FOR AUSTRALIA ANTIGEN AND ANTIBODY THERETO [75] Inventors: Girish N. Vyas, San Francisco,

Calif.; Nahum Raphael Shulman, Kensington, Md.

[73] Assignee: The United States of America as represented by the Secretary of the Department of Health, Education & Welfare, Washington, DC.

[22] Filed: Aug. 9, 1971 [2]] Appl. No.: 170,292

[52] US. Cl 424/12; 260/112 R; 260/112 B;

424/3; 424/8 [51] Int. Cl.... Cl2k 1/00;G01n 31/00; GOln 33/16 [58] Field of Search 424/12; 260/112 R, 112 B [56] References Cited OTHER PUBLICATIONS LeBouvier, Advances in Virus Res. Vol. 16, 1970, pp. 357-396.

Shulman, The Amer. J. Med. Vol. 49, Nov. 1970, pp. 669-692.

Atlas Powder Co., Catalog of Products for Cosmetic Formulations, Jan. 1, 1957, pp. 6-7.

.luji, Japan. J. Exp. Med. Vol. 39, 1969, pp. 615-620.

Vyas (2), Blood, Vol. 34, Nov. 1969, pp. 573-581. Barker, .1. Immuno, Vol. 102, 1969, pp. 1529-1532. Alter, Blood, Vol. 27, 1968, pp. 297-309.

Bolin, AJCP, Vol. 49, 1968, pp. 635-646.

Kabat, Exper. Immunochem. cc Thomas, Springfield, 111., 2nd Ed. 1961, pp. 97-101, 113-125.

Baker, Applied Microbiol., Vol. 17, March, 1969, pp. 422-426.

Vyas, .1. of lmmunol. Vol. 100, No. 2, 1968, pp. 274-279.

Primary Examiner-Albert T. Meyers Assistant Examiner-A. P. Fagelson 5 7] ABSTRACT 8 Claims, No Drawings HEMAGGLUTINATION METHOD FOR AUSTRALIA ANTIGEN AND ANTIBODY THERE'IO BACKGROUND OF THE INVENTION The invention relates to detecting the agent of viral hepatitis and antibodies against it, and more particularly to the diagnosis or hepatitis or determining previous encounters with hepatitis by the quantitive determination of hepatitis-associatedantigen or antibody in serum from a patient.

Viral hepatitis is a relatively common disease. but the disease is sometimes difficult to diagnose and there is, as yet, no specific treatment. Hepatitis poses a serious problem in detecting blood donors who may transmit the disease. Even though as many 5% of the reported cases become chronically ill and another 2% become cirrhotic, it has been estimated that three out of every thousand persons who become infected with the serum hepatitis virus do not become ill, but nevertheless carry the disease.

The known methods for detecting viral hepatitis, rely on detection of virus products in the body fluids but are time-consuming and burdensome as well as being relatively insensitive. In fact, it has been estimated that most of the prior art tests can detect only to of the carriers of the disease. Moreover, prior tests have not been sensitive enough to detect antibodies that develop after a single exposure to hepatitis virus or primary hepatitis infection. A test sensitive enough to detect antibodies arising under these circumstances is essential to evaluate immunity and potential effectiveness of vaccines.

A specific antigen, popularly known as Australia antigen, has been found in the serum of many patients with viral hepatitis. The exact pathogenic role of the Australia antigen is a current problem of great academic interest as well as urgency in view of the importance of recognizing the source and/or cause of hepatitis. The Australia antigen is also known as hepatitisassociated-antigen, and both terms will be used interchangeably herein. Most of the common methods used for the detection of viral hepatitis rely on the presence of the Australia antigen, or the antibody thereto, for their specificity.

At present the neutralizing effect of immune serum, that is, the serum of an individual who has recovered from viral hepatitis, and contains antibodies specific against the disease. is used to diagnose heptatitis by the so-called agar-gel precipitation test. In this test an antibody and the sample are added to agar-gel and diffuse to form a precipitate of antibody-antigen complex if the hepatitis-associated-antigen is present in the sample. This test. however, is not very sensitive, requires long periods of time to react, and does not permit accurate quantitative determination of the antigen present in the sample.

Other techniques exist for detecting and diagnosing hepatitis, such techniques including immunodiffusion, complement fixation, and electrophoretic modifications of precipitin techniques are the simplest but the least sensitive and slowest. The electrophoretic modifications of precipitin techniques are rapid, being as rapid as complement fixation, but are relatively insensitive. Of the prior art techniques. complement fixation procedures appear to be the most sensitive and rapid. The complement fixation technique involves adding a biologic material such as blood or plasma to the appropriate antibody for hepatitis-associatedantigen. The reaction mixture is incubated with a predetermined amount of complement to fix the latter. By titrating the amount of remaining non-fixed complement by means of incubation with a standardized cell solution, the amount of hepatitis-associated-antigen or antibody present in the body fluid can be determined. While the complement fixation technique is sensitive for detecting antigen it is not very sensitive for detecting antibody and is not available as a routine technique in many hospitals and blood banks.

Accordingly, it is an object of the present invention to provide a method for diagnosing viral hepatitis and detecting carriers thereof which is free of the aforementioned and other such disadvantages.

It is a primary object of the present invention to provide a method for diagnosing viral hepatitis and detecting carriers thereof by the qualitative or quantitative determination of hepatitis-associated-antigen or antibody using standardized reagents which can be preserved over a period of time.

It is another primary object of the present invention to provide a method for diagnosing viral hepatitis and detecting carricrs thereof which is simple, sensitive, specific, and rapid.

It is another object of the present invention to provide a method for diagnosing viral hepatitis and detecting carriers thereof by the qualitative or quantitative determination of hepatitis-associated-antigen by hemagglutination-inhibition.

It is yet another object of the present invention to provide a sensitive method for diagnosing viral hepatitis and detecting carriers thereof as well as individuals who are potentially immune to hepatitis by the qualitative or quantitative determination of hepatitisassociated antibody by hemagglutination.

It is yet another object of the present invention, consistent with the foregoing objects, to provide a method for the determination of hepatitis-associated-antigen or antibody in serum which includes coating inert indicator red blood cells with purified hepatitis-associatedantigen, contacting the coated cells with test serum, and detecting the presence or absence of hemagglutination.

SUMMARY OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION The hemagglutination method of the present invention is at least as sensitive and rapid as the complement fixation technique for detecting hepatitis-associatedantigen. and is easy to perform with reagents that can be standardized for use over long periods. The technique of the present invention is more sensitive than complement fixation and approximately as sensitive as radioimmunoassay for detecting antibody to hepatitisassociated-antigen. The agglutinogen consists of inert indicator red cells coated with isolated hepatitisassociatedantigen with chromic chloride as a coupling agent. The agglutinator is antibody to the antigen.

The standard antiserum to hepatitis-associatedantigen used as an agglutinator was derived from a hemophiliae who had received several units of plasma and had produced antibodies specific for the antigen.

EXAMPLE 1 Plasma samples from asymptomatic carriers of hepatitis-associatedantigen where used to isolate purified antigen. Isolation was carried out by combined rate and isopycnic zonal centrifugation with cessium chloride density gradients. The purified preparations were dialyzed extensively against 0.85% sodium chloride (saline) at 4 C. The Kjeldahl nitrogen content of the purified antigen used was 170 pug/m1, and the optical density at 280 nm wavelength was 0.445. This antigen preparation gave a single band in agarose at a maximum dilution of 1:2 with antiserum to the antigen, and when concentrated tenfold gave no precipitin band with rabbit antiserum against whole human plasma.

1 Anderson et al.. Nat. Cancer [1151. Mmwgiz. 21. 253 (1966).

2 Barker et al.. J. ImImmuL. 102. I529 (1969). Vyas and Shulman.

Science 170. 332 (I970) EXAMPLE II Indicator cells were prepared from group human blood collected in ethylenediaminetetraacetate (EDTA). Cells were stored at 4 C. for no more than a week prior to being coated with the antigen. Red cells were washed four times with saline and used as a 40% suspension in saline. To coat the cells. 0.025 ml of cell suspension was mixed with 0.075 ml of purified antigen, and 0.025 ml of 1.25 mM chromic chloride was added. The mixture was agitated gently in a glass tube by 75 mm) at room temperature for 5 minutes. Periods up to minutes could be used without modifying results. The cells were then washed four times with saline and used as a 0.2% suspension in phosphatebuffered saline, pH 7.3. containing 0.5% bovine serum albumin, 0.002570 polyvinyl pyrrolidone, and a 1:20.000 dilution of Tween 80 (polyoxyethylene sorbitan monooleate in the form of an oily liquid of specific gravity 1.05-1.10, manufactured by Atlas Chemical 1ndustries, Inc., Wilmington, Delaware). For the antigen preparation used these conditions of coating were found to be optimum by a checkerboard test with different dilutions of antigen and chromic chloride. However. the optimal concentration of chromic chloride has been found to vary several fold depending on the purity and abundance of antigen in any one specific preparation. The purity and abundance of antigen of a specific preparation can be determined and the optimum concentration of chromic chloride easily calculated by one or ordinary skill in the art.

' \'as et al.. .I. IMMINOL. I00. 274 (1968) EXAMPLE Ill Hemagglutination reactions for detection and titration of antibodies were carried out in V-shaped microtiter platesv 0.025 ml of cells coated with the antigen was added to 0.025 ml of serial twofold dilutions of serum made with the Tween. bovine serum albumin. polyvinyl pyrrolidone buffer. After incubating these reactions for one hour at room temperature the micro titer plates were centrifuged at 1,200 revolutions per minute for approximately 30 seconds. and the plates were kept at an angle of 60 for 15 to 20 minutes before they were read. A discrete of agglutinated cells was seen as a dot. and a smooth line of red cells which streamed down the vertex of the V-shaped well in the microtiter plate consisted of unagglutinated cells. The antibody titer with the highest dilution of serum showing definite agglutination.

The specificity of antibody to the antigen was established by hemagglutination inhibition with purified antigen and known antigen-positive serums and failure to inhibit agglutination with known hepatitis-associatedantigen-negative serums.

A total of 72 serums was tested for antibodies by compliment fixation (CF), immuno diffusion (ID). and hemagglutination (HA). The results are shown in Table I.

TABLE I Antibody assay on 72 serums by HA. CF & ID. HA titers are reciprocal to solutions of serum showing agglutination.

No. of HA Number of serums serums titers positive in CF ID EXAMPLE IV To test for hepatitis-associated-antigen in serum samples. 0.025 ml of test serum at 2-, 4-. 8-, and 16-fold dilutions were mixed with 0.025 ml of antibody to the antigen containing 10 agglutinating units. defined as 10 times the minimum amount of antibody that gave a positive agglutination. 0.025 ml of cells coated with the antigen was then added. As a control. a 1:2 dilution of each serum was tested with antigen-coated cells in the absence of antibody to the antigen. In all. 523 serum samples obtained from patients during the incubation period. acute phase. and convalescent phase if viral hepatitis were tested by hemagglutination inhibition. and the results are compared in Table 2 with those of CF and ID performed previously and reported. The test for hemagglutination inhibition was read as follows: partial inhibition of agglutination in 1:2 dilution was considered negative. definite inhibition of agglutination in 1:2 dilution was a weak but definitely positive test for the presence ofthe antigen. inhibition of agglutination in 1:4 and higher dilutions was a more strongly positive test for the antigen.

' Shulman el :11. :IHH. In]. .WnL. 72. 257 I970).

TA BLE 2 Results Assay Nega- Weak Positi\'e positive the ID 463 60 CF 326 55 142 HA1 321 60 142 As can be seen in Table 2. the sensitivity of the CF and hemagglutination inhibition are similar, whereas the ID test is much less sensitive. Of the 142 positive results with CF, 29 specimens were strongly anticomplementary, indicating the presence of large amounts of antigen-antibody complexes. 5 samples of another patient that where negative in CF where positive in hemagglutination inhibition at a dilution of 1:2. The hemagglutination inhibition tests can be applied with reagents that can be stored for at least a week and longer. The test also lends itself to automation.

While there is shown and described a specific embodiment of the present invention it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims. ACCORD- lNGLY,

What is claimed is:

l. A method for measuring Australia antigen in serum or other biological material comprising coating inert indicator human red blood cells with purified Australia antigen using chromic chloride as a coupling agent, suspending said coated cells in phosphate buffered saline solution containing bovine serum albumin, polyvinyl pyrrolidone, and polyoxyethylene sorbitan monooleate, adding an aliquot of a predetermined dilution of antibody to said antigen to each of serial twofold dilutions of said serum or other material, and adding said suspension of coated cells to said serumantibody mixture, whereby inhibition of hemagglutination in 1:2 dilution and higher dilutions indicates the presence of said antigen in said serum.

2. The method of claim 1, wherein said phosphate buffered saline solution is at a pH of 7.3, and contains 0.5 percent bovine serum albumin, 0.0025 percent polyvinyl pyrrolidone, and a l:20,000 dilution of polyoxyethylene sorbitan monooleate in the form of an oily liquid of specific gravity l05-l.10.

3. The method of claim 1, wherein said red blood cells are coated with said purified antigen by suspending said cells as a 40 percent suspension in saline solution, mixing said cell suspension of cells in saline solution with three times the volume of purified antigen. adding an amount of 1.25 mM chromic chloride equal to the original volume of said cell suspension, agitating the resulting mixture for 5-20 minutes. washing the thus coated cells and suspending said coated cells in 6 said phosphate buffered saline solution.

4. The method of claim 1, wherein said purified antigen is obtained by isolating antigen from plasma samples by combined rate and isopycnic zonal centrifugation with cesium chloride density gradients. and dialyzing against 0.85% saline solution at 4C, said purified antigen having a Kjeldahl nitrogen content of ug/ml and an optical density at 280 nm wave length of 0.445, said purified antigen further giving a single band in agarose at a maximum dilution of 1:2 with antiserum to the antigen and, when concentrated tenfold gives no precipitin band with rabbit antiserum against whole human plasma.

5. A method for measuring antibody to Australia antigen in serum or other biological fluid comprising coating inert indicator human red blood cells with purified Australia antigen using chromic chloride as a coupling agent, suspending said coated cells in phosphate buffered saline solution containing bovine serum albumin, polyvinyl pyrrolidone, and polyoxyethylene sorbitan monooleate, and adding an aliquot of a predetermined dilution ofsaid coated cells to each of serial twofold dilutions of said serum or fluid in a microtiter plate, incubating the resulting mixtures, and centrifuging said microtiter plates whereby hemagglutination in serums with titers of at least 4 indicates the presence of said antibody in said serum.

6. The method of claim 5, wherein said phosphate buffered saline solution is at a pH of 7.3, and contains 0.5 per cent bovine serum albumin, 0.0025 percent polyvinyl pyrrolidone, and a l:20,000 dilution of polyoxyethylene sorbitan monooleate in the form of an oily liquid of specific gravity 1.05-1.10.

7. The method of claim 5, wherein said red blood cells are coated with said purified antigen by suspending said cells as a 40 percent suspension in saline solution. mixing said cell suspension of cells in saline solution with three times the volume of purified antigen, adding an amount of 1.25 mM chromic chloride equal to the original volume of said cell suspension, agitating the resulting mixture for 5-20 minutes, washing the thus coated cells and suspending said coated cells in said phosphate buffered saline solution.

8. The method of claim 5, wherein said purified antigen is obtained by isolating antigen from plasma samples by combined rate and isopycnic zonal centrifugation with cesium chloride density' gradients, and dialyzing against 0.85% nitrogen solution at 4C, said purified antigen having a Kjeldahl nitrogen content of 170 M ml and an optical density at 280 nm wave length of 0.445. said purified antigen further giving a single band in agarose at a maximum dilution of 1:2 with antiserum to the antigen and. when concentrated tenfold gives no precipitin band with rabbit antiserum against whole human plasma. 

1. A method for measuring Australia antigen in serum or other biological material comprising coating inert indicator human red blood cells with purified Australia antigen using chromic chloride as a coupling agent, suspending said coated cells in phosphate buffered saline solution containing bovine serum albumin, polyvinyl pyrrolidone, and polyoxyethylene sorbitan monooleate, adding an aliquot of a predetermined dilution of antibody to said antigen to each of serial two-fold dilutions of said serum or other material, and adding said suspension of coated cells to said serum-antibody mixture, whereby inhibition of hemagglutination in 1:2 dilution and higher dilutions indicates the presence of said antigen in said serum.
 1. A METHOD FOR MEASURING AUSTRALIA ANTIGEN IN A SERUM OR OTHER BIOLOGICAL MATERIAL COMPRISING COATING INERT INDICATOR HUMAN RED BLOOD CELLS WITH PURIFIED AUSTRALIA ANTIGEN USING CHROMIC CHLORIDE AS A COUPLING AGENT, SUSPENDING SAID COATED CELLS IN PHOSPHATE BUFFERED SALINE SOLUTION CONTAINING BOVINE SERUM ALBUMIN, POLYVINYL PYRROLIDONE, AND POLYOXYETHYLENE SORBITAN MONOOLEATE, ADDING AN ALIQUOT OF A PREDETERMINED DILUTION OF ANTIBODY TO SAID ANTIGEN TO EACH OF SERIAL TWO-FOLD DILUTIONS OF SAID SERUM OR OTHER MATERIAL, AND ADDING SAID SUSPENSION OF COATED CELLS TO SAID SERUM-ANTIBODY MIXTURE, WHEREBY INHIBITION OF HEMAGGLUTINATION IN 1:2 DILUTION AND HIGHER DILUTIONS INDICATES THE PRESENCE OF SAID ANTIGEN IN SAID SERUM.
 2. The method of claim 1, wherein said phosphate buffered saline solution is at a pH of 7.3, and contains 0.5 percent bovine serum albumin, 0.0025 percent polyvinyl pyrrolidone, and a 1:20,000 dilution of polyoxyethylene sorbitan monooleate in the form of an oily liquid of specific gravity 105-1.10.
 3. The method of claim 1, wherein said red blood cells are coated with said purified antigen by suspending said cells as a 40 percent suspension in saline solution, mixing said cell suspension of cells in saline solution with three times the volume of purified antigen, adding an amount of 1.25 mM chromic chloride equal to the original volume of said cell suspension, agitating the resulting mixture for 5-20 minutes, washing the thus coated cells and suspending said coated cells in said phosphate buffered saline solution.
 4. The method of claim 1, wherein said purified antigen is obtained by isolating antigen from plasma samples by combined rate and isopycnic zonal centrifugation with cesium chloride density gradients, and dialyzing against 0.85% saline solution at 4*C, said purified antigen having a Kjeldahl nitrogen content of 170 Mu g/ml and an optical density at 280 nm wave length of 0.445, said purified antigen further giving a single band in agarose at a maximum dilution of 1:2 with antiserum to the antigen and, when concentrated tenfold gives no precipitin band with rabbit antiserum against whole human plasma.
 5. A method for measuring antibody to Australia antigen in serum or other biological fluid comprising coating inert indicator human red blood cells with purified Australia antigen using chromic chloride as a coupling agent, suspending said coated cells in phosphate buffered saline solution containing bovine serum albumin, polyvinyl pyrrolidone, and polyoxyethylene sorbitan monooleate, and adding an aliquot of a predetermined dilution of said coated cells to each of serial two-fold dilutions of said serum or fluid in a microtiter plate, incubating the resulting mixtures, and centrifuging said microtiter plates whereby hemagglutination in serums with titers of at least 4 indicates the presence of said antibody in said serum.
 6. The method of claim 5, wherein said phosphate buffered saline solution is at a pH of 7.3, and contains 0.5 per cent bovine serum albumin, 0.0025 percent polyvinyl pyrrolidone, and a 1:20, 000 dilution of polyoxyethylene sorbitan monooleate in the Form of an oily liquid of specific gravity 1.05-1.10.
 7. The method of claim 5, wherein said red blood cells are coated with said purified antigen by suspending said cells as a 40 percent suspension in saline solution, mixing said cell suspension of cells in saline solution with three times the volume of purified antigen, adding an amount of 1.25 mM chromic chloride equal to the original volume of said cell suspension, agitating the resulting mixture for 5-20 minutes, washing the thus coated cells and suspending said coated cells in said phosphate buffered saline solution. 